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In Vitro Determination of Marine Spon Effect Against Human Breas Ination Available online at http://www.journalcra.com INTERNATIONAL JOURNAL OF CURRENT RESEARCH International Journal of Current Research Vol. 5, Issue, 02, pp. 124-128, February, 2013 ISSN: 0975-833X RESEARCH ARTICLE In vitro Determination of Marine Sponge Hyrtios erectus Secondary Metabolite Effect against Human Breast and Larynx Cancer Cell Lines 1Ramachandran, M. 2Titus Immanuel, 1Manley Backyavathy, P. and 1*Balwin Nambikkairaj 1Department of Zoology, Voorhees College, Thiruvalluvar University, Vellore, Tamil Nadu, India 2Fisheries Division, Central Agricultural Research Institute, Port Blair, A and N Islands, India ARTICLE INFO ABSTRACT Article History: Marine sponges are rich sources of novel secondary metabolite and they are potential drug molecule to antitumor Received 19th November, 2012 and antiprolific drug development. Hyrtios erectus was extracted and purified through different solvent fractions Received in revised form methods. Pure compound was obtained as brownish amorphous powder. The positive test for dragendorff reagent 24th December, 2012 was indicated as alkaloid group. Cytotoxicity was tested on Normal (Vero) cell line non tumor cells, Human Accepted 17th January, 2013 Breast cancer cell line (MCF-7) and Human epithelial larynx cancer cell line (Hep-2) using microculture th tetrazolium (MTT) assay for anticancer activity. Sponge pure compound was non‐toxic to (Vero) cells but highly Published online 14 February, 2013 cytotoxicity to (53%) MCF-7 cells and low toxic to Hep-2(32%) was recorded at 25μg/ml concentration. Each concentrations express individual results of cells viability with cytotoxicity increase and decrease of their Key words: concentration level of pure compound. In this research further investigation require for this sponge purified Hyrtios erectus, compound in order to chemical structure elucidations as well as pre clinical study for anticancer activity. Antitumor, Cytotoxicity, Methanol extract, Zoochemical screening. Copy Right, IJCR, 2013, Academic Journals. All rights reserved. INTRODUCTION different Hyrtios sp. and their associated microorganisms revealed the presence of numerous structurally unique natural products Marine sponge are rich source of structurally novel and biologically including scalarane sesterterpenes, acyclic triterpenes, indole active secondary metabolites considered to be true “chemical alkaloids and macrolides in addition to steroids. Many of these factories” producing hundreds of unique chemical compounds with compounds possess interesting biological activities. spongistatins, the different biological activities have been isolated. Sponges are simple, most important metabolites of the genus Hyrtios discovered to date, multicellular, sessile invertebrates with no true tissue layers or organ; showed powerful anticancer activity (Youssef, 2002; Pettit, 1998). they are cytotoxic, inhibit cell proliferation and could be used as The chemical investigations, which afforded the antineoplastic agents chemotherapeutics (Bergquist, 1978). These compounds differ sesterstatins (Longeon et al., 2011; Pettit et al., 1998). Cytotoxicity structurally and act on different cytoskeleton elements, but have assays are a widely used method in vitro toxicology studies. It is not similar anti prolific and antitumor activities. Moreover, many only rapid and standardized, but also a sensitive and inexpensive antitumor drug isolated and their structure determined, but their method to measure drug induced alterations in metabolic pathways or biological roles and activities are still largely unknown (Bacus and structural integrity which may or may not be related to directly to cell Green, 1974; Kornprobst,2005). Hyrtios erectus synonym H.erecta death (Pettit and Tan, 2005; Habib and Karim, 2011). Recent (taxonomy: phylum Porifera, class Demospongiae, order developments in the field of marine antitumor research with emphasis Dictyoceratida, family Thorectidae) is an animal colonies, sedentary, on the signal transduction pathways of oncogenesis, clinical status of grayish in color, irregularly rounded, asymmetrical, attached to sea the marine derived antitumor compounds and discuss the potential bottom by means of masses of spicules. challenges and probable strategies to combat the gap between marine compound isolation and successful antitumor drug development. These colonies are attached on the dead coral stones in shallow water inter-tidal areas at the depth of 10-20m. Sponge secondary metabolites show interesting biological activities, for example MATERIALS AND METHODS calyculins from Discodermia calyx (Sipkema et al., 2005), discodermolide from D. dissolute (Kato et al., 1986), latrunculins Sponge collection from Latrunculia magnifica (Gunasekera, 1990; Kashman, 1980; Kashman et al., 1985; Spector, 1983), and spongistatins from Sponge tissue was collected by scuba divers at (10-20m) deep along Spongia sp. and Spirastrella sp. (Spector, 1989; Petit, 1993). The the South Andaman Sea coast (North Bay and Pongibalu secondary metabolites from the genus Hyrtios sp particularly region11°30’N and 92°39’E). The sponge collection was made in the Hertios erectus synonym H.erecta have been investigated extensively month of June, 2011. Samples were kept instead plastic packs in ice (Petit et al., 1994; Kobayashi et al., 1994; Miyaoka, 2000; Pettit boxes before freezing at -20°C until analysis. Sample was preserved et al., 1998; Yonghong, 2007). Previous chemical investigations of in 100% methanol solvent. Taxonomy details were identified and deposited at the Central agricultural research institute, Fisheries *Corresponding author: [email protected] division, Port Blair. A voucher specimen is under the accession number (PB5985). 125 International Journal of Current Research, Vol. 5, Issue, 02, pp. 124-128, February, 2013 Extraction/Fractionation/Isolation method of plots of the percent of cell viability against the concentration of the tested compounds. The fresh sponge Hyrtios erectus (3kg weight) was chopped into small pieces and filled in glass containers soaked in methanol RESULTS (3 x 2.5L) which was shacked well twice daily around one weak. Methanol extract was filtered by No.1 Whatman filter paper (GE The pure compound was isolated from marine sponge ‘hexane’ with Healthcare, 125mm, UK) pooled into rotary evaporator (STRIKE ‘chloroform’ combined fraction and brownish amorphous powder 202, Germany). Methanol extract was concentrated under reduced o respectively. The positive tests of the pure compound are determined pressure 45 C, which was further dried under high vacuum (42g). by the method of dragendorff reagent which was identified alkaloid The residual mass was further extracted with methanol-chloroform group. In this research three cancer cell lines are (Vero) normal cell (80:20%). Complete drying the methanol-chloroform extract, a line non tumor cells, (MCF-7) Human Breast cancer cell line and residual mass of (23g) was obtained. Methanol extract showing (Hep-2) Human epithelial larynx cancer cell line selected for promising activity in vitro models, it was fractionated into four anticancer assays. In this study, the normal Vero cell line expressed fractions, i.e. the hexane(2g), chloroform(4g), n-butanol (6g)soluble 91% cells viability and 8% of inhibition of cancer cells was found at and n-butanol (11g) insoluble fractions. The hexane fraction was low concentration 25mg/ml of pure compound are shown in Table.1. found to be a complex mixture and was of low yield. Moreover, The (MCF-7) breast cancer cell line low viability 46% and cytotoxic hexane and chloroform fractions were mixed combined fraction was 53% was observed, even though 14% of viability and 85% of included for purification of its components was achieved by cytotoxic in 500mg/ml concentration level was recorded (Fig.1-2). chromatography over column of silica gel. One pure compound was Similarly result of (Hep-2) cancer cell line cells viability 67% and obtained after re-chromatography over flash silica gel (230-400 cytotoxicity 32% was obtained at 25mg/ml concentration. Low mesh) of the combined chloroform and hexane fraction (Lakshmi et percentage of viability 20% and high cytotoxicity 70% was found at al., 2012). Thin Layer Chromatography (TLC) analysis was carried 500mg/ml concentration on (Hep-2) cell line. The pure compound out for identifying purified compound spot using aluminium sheet was expressed potent cytotoxic activity 53% at 25mg/ml against precoated silica gel 60 F254 or on glass precoated RP-18 F254 plates (MCF-7) cancer cell line than Hep-2 cancer cell line. Minimum (Merck, Darmstadt, Germany). effective concentration of pure compound of sponge was non‐toxic to (Vero) cells but highly toxic to 53% (MCF-7) cells and low toxic Zoo chemical analysis to 32% (Hep-2) cells were recorded(IC50) at 25μg/ml concentration of the pure compound (Fig. 3-4). Pure compound was highly potent The Zoochemical analysis absence of an established protocol for the effect on cervical cancer cells could be inhibit on the cells screening of chemical constituents in animal tissues, the qualitative proliferative activity. Similarly increase the dose could increase the analysis for the possible bioactive components present in the sponge activity on cancer cells is dose dependent manner. In the presence of samples were done using established phytochemical screening 400 mg concentration of compound titrated on cancer cells protocol (Edeoga , 2005). contribute low percentage cells viability and high Cytotoxicity Evaluation The cell lines
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